The present invention relates to the field of immunoassay techniques for the detection and determination of immunologically active substances in biological specimens and in particular to a novel test system and procedure for performing quantitative immunoassays and to processes for preparing the test system and carrying out the procedure.
A number of different types of immunoassays are currently in use for the detection, identification and quantitation of immunologically determinable substances, e.g. antigens and antibodies, in various biological materials. In carrying out an immunoassay, a labeled, immunoreactive substance is employed either directly or conjugate, by means of an immune reaction between the two substances. Even substances that are not immunogenic by themselves, such as haptens, can be determined by immunoassay if they are bound to a macromolecular carrier capable of inducing antibody to the lower molecular weight substance. Immunoassays are useful in determining the occurrence of immune reactions in blood serum, e.g. for monitoring and/or detecting infectious diseases, and are also widely used in a number of immunohistochemical methods performed on tissue. Recent advances in immunoassay techniques involving in vitro testing of minute levels of host antibody response to an infectious agent have become the method of choice for the detection of infectious disease.
Immunoassays may be carried-out either in solution, or on various solid supports to which the immunologically determinable substance or its immunospecific conjugate is affixed. As between the two, the latter procedure, known as solid-phase immunoassay, is preferred in practice because of its simplicity of performance and the ease with which the immune complexes formed on the solid phase may be separated from unreacted antigen or antibody.
One commonly used type of solid-phase. immunoassay involves a non-competitive binding technique for determination of antigen or antibody. In performing an immunoassay of this type for the determination of antibody, antigen is immobilized on a solid support and then contacted with the test specimen suspected of containing the antibody of interest. Thereafter, a second labeled antibody that reacts specifically with the antibody of interest is added to the sample. Because the second antibody is labeled, the presence of the antibody of interest can be determined. By means of this technique, commonly known as the "sandwich" or "second antibody" technique, specific antibody can readily be detected in blood serum, for example. The radioallergosorbent test (RAST) for the measurement of specific IgE antibody to a variety of allergens is based on this procedure, as are assays for the measurement of antibodies to a number of infectious agents, such as hepatitis B, rubella, influenza and human T-cell lymphotropic virus III (HTLV III), the virus apparently responsible for Acquired Immune Deficiency Syndrome (AIDS).
In certain cases, the "sandwich" technique may be modified to include one or more additional layers of antigen or antibody to increase the sensitivity of the assay, or to permit the detection of antibody to antigens that are not easily attached to a solid surface.
Solid-phase immunoassays are also performed as competitive binding assays, based upon competition between labeled and unlabeled forms of an immunologically determinable substance for binding sites on the immunospecific conjugate. For example, in order to determine the amount of antibody present in a biological specimen using a competitive assay, a test sample is mixed with labeled antibody, and then contacted with the corresponding antigen bound to a solid support, with any unlabeled antibody present in the sample competing with labeled antibody for antigen binding sites or determinants. The test sample is then separated into a liquid-phase and a solid-phase and the relative amount of labeled antibody present in either phase is determined. Most competitive binding assays operate on the principle that any immunologically determinable substance present in the test sample and a labeled form of the same substance will be attracted with essentially equal affinity to a solid support bearing the immunospecific conjugate. Therefore, the labeled and unlabeled forms of the immunologically determinable substance become bound to the support in amounts proportional to the relative amounts of each substance present in the test sample.
Radioactively labeled reagents have been employed for some time in the above-described immunoassays, because of the sensitivity and accuracy of the assays employing such reagents. The radioisotope labels provide a readily measurable signal which permits the results of the assay to be determined directly. More recently, enzyme-labeled reagents have been widely used in immunoassays. In performing an immunoassay using an enzyme-labeled reagent, the enzyme activity of one of the separated portions of the test sample is measured by incubating the appropriate fraction with a substrate for producing an enzyme-catalyzed reaction, typically involving production or extinction of a colored compound whose adsorption may be readily measured either qualitatively or quantitatively. Such assays are reported to be comparable to the radioassays in sensitivity and offer certain operational advantages over radioimmunoassay techniques. Various chromophoric materials, such as fluorescent or phosphorescent substances are also commonly employed as labels in the above-described immunoassays.
If quantitation of an immunologically determinable substance is desired in carrying out an immunoassay, a standard curve is constructed from data gathered from a pre-determined number of samples, each containing the same known concentration of a labeled form of the immunologically determinable substance and various known concentrations of unlabeled immunospecific conjugate. The samples are incubated, any free immunologically determinable substance is separated from that which is bound or complexed with the immunospecific conjugate, and then, by use of an appropriate detector, the percent of either the free or bound labeled immunologically determinable substance, or both, is determined. This procedure is repeated for all of the samples and the percent of bound labeled immunologically determinable substance is plotted as a function of the concentration of immunologically determinable substance. Typically, as the total concentration of immunologically determinable substance increases, the relative amount of the labeled form thereof bound to the immunoreactive conjugate decreases. Once the standard curve is prepared, it is thereafter used to determine the concentration of immunologically determinable substance of interest in test samples undergoing analysis.
In the above-described solid phase immunoassays, for each immunologically determinable substance sought to be determined, the corresponding immunospecific conjugate, in purified form, is required for preparation of the solid phase. Thus, each solid phase reagent can be used for detection of a single immunoreactive substance, and for no other substance. Furthermore, the methodology involved in the prior art immunoassays generally requires two or more adsorption steps, each of which entails washing of unbound immunoreactive substance and incubation. The number and nature of the process steps employed in the immunoassays of the prior art tend to adversely affect the quantitative reliability of the results obtained thereby.
The development of an immunoassay for the determination of immunologically determinable substances in a more efficient manner with greater quantitative reliability than the procedures heretofore available remains a highly desirable objective. This is particularly true with respect to the detection and/or monitoring of infectious diseases based on a determination of a host's production of antibody in response to the infectious agent.